Stable factor VIII/von Willebrand factor complex

ABSTRACT

There are disclosed a stable factor VIII/vWF-complex, particularly comprising high-molecular vWF multimers, being free from low-molecular vWF molecules and from proteolytic vWF degradation products, as well as a method of producing this complex.

The invention relates to a stable virus-safe factor VIII-complex,particularly comprising high-molecular vWF multimers of high structuralintegrity and free from low-molecular vWF molecules and from proteolyticvWF degradation products. Furthermore, the invention relates to a methodof recovering and producing a stable factor VIII complex as well aspharmaceutical preparations thereof.

The coagulation of blood is a complex process including the sequentialinteraction of a number of components, in particular of fibrinogen,factor II, factor V, factor VII, factor VIII, factor IX, factor X,factor XI and factor XII. The loss of one of these components or theinhibition of its functionality leads to an increased tendency ofhemorrhaging which may be life-threatening for some patients.

Von Willebrand factor (vWF) circulates in plasma complexed with factorVIII, factor VIII aiding the coagulation of blood and vWF in the complexwith factor VIII stabilizing the latter and protecting it fromproteolytic degradation. By its function in platelet aggregation, vWFalso directly interferes in the coagulation of blood. vWF is aglycoprotein formed in different mammalian cells and subsequentlyreleased into circulation. Starting from a polypeptide chain having amolecular weight of approximately 220 kD, a vWF dimer having a molecularweight of 550 kD is formed in the cells by formation of several sulfurbonds. From the vWF dimers, further polymers of vWF of ever increasingmolecular weights, up to 20 million Daltons, are formed by linkage.Therefore, vWF exists in plasma in a series of multimer forms havingmolecular weights of from 1×10⁶ to 20×10⁶ Daltons. It is assumed thatparticularly the high-molecular vWF multimers are of essentialimportance in the coagulation of blood.

Besides the carrier function for coagulation factor VIII, vWF has thefunctions of bridge formation between vessel wall and the platelets andof platelet agglutination. The basis for platelet agglutination is givenby the binding of vWF to surface receptors (glycoproteins Ib, IIb/IIIa).The binding site within vWF for binding to GP Ib is located in disulfideloop Cys(509)-Cys(695). It is known that platelet agglutination startswith the binding of vWF to glycoprotein Ib. Following an activationsignal, binding of vWF to the glycoprotein IIb/IIIa-complex andagglutination occur. Binding of vWF to the surface receptors thus is aprerequisite for platelet agglutination; the binding of severalplatelets by a vWF molecule leads to agglutination. vWF-platelet bindingthus constitutes the molecular cause for platelet agglutination.

In hemophilia, blood coagulation is disturbed by a deficiency of certainplasmatic blood coagulation factors. In hemophilia A, the tendency tohemorrhage is based on a deficiency of factor VIII or on a deficiency ofvWF, respectively, which is an essential component of factor VIII.Treatment of hemophila A primarily is effected by replacing the lackingcoagulation factor by factor concentrates, e.g. by infusion of factorVIII, factor VIII-complex or vWF.

vWF Syndrome has several clinical pictures which go back to anunderproduction or overproduction of vWF. Thus, e.g., an overproductionof vWF leads to an increased thrombosis tendency, whereas an undersupplyis caused by the absence or reduction of high-molecular forms of vWFwhich manifests itself by an increased hemorrhage tendency and anextended hemorrhaging period due to an inhibited platelet aggregation.The deficiency of vWF may also cause a phenotypical hemophila A, sincevWF is an essential component of functional factor VIII. In theseinstances, the half-life of factor VIII is reduced so much that itsfunction in the blood coagulation cascade is impaired. Patientssuffering from von Willebrand disease (vWD) thus frequently exhibit afactor VIII deficiency. In these patients, the reduced factor VIIIactivity is not the consequence of a defect of the X-chromosomal gene,but is an indirect consequence of the quantitative and qualitativechange of vWF in plasma. The differentiation between hemophilia A andvWF normally can be effected by measuring vWF antigen or by determiningthe ristocetin cofactor activity. Both, the vWF antigen content and theristocetin cofactor activity is lowered in most vWD patients, whereas itis normal in hemophilia A patients.

Conventional methods for the therapy of von Willebrand syndrome are withvWF recovered from plasma, and there exists a number of suggestions totreat vWD patients with purified vWF or with factor VIII/vWF-complex.

Purification of factor VIII or of factor VIII-complex from plasma orfrom cryoprecipitate is even more difficult because factor VIII ispresent in plasma in very small amounts only, is extremely unstable, andthe association of factor VIII with vWF is reversible under specificconditions. Factor VIII is recovered from plasma by purification andconcentration, yet, depending on the purification method, instabilityand loss of factor VIII activity may occur because vWF and factor VIIIare separated during purification. Thus, the final product frequently isa mixture of stable factor VIII-complex and unstable factor VIII, aswell as of contaminating proteins, such as, e.g., fibrinogen,fibronectin or vitamin K-dependent proteins which could not be removedby the purification. Because of the instability of the purified complex,stabilizers, such as albumin or amino acids etc., are admixed. However,the presence of contaminating proteins and/or stabilizers in thepurified product did reduce the specific activity of the factorVIII-complex.

EP 0 468 181 describes a method of purifying factor VIII from humanplasma by ion exchange chromatography, elution of factor VIII with highionic strength at acidic pH and collecting the eluate in the presence ofa stabilizer, such as heparin, albumin and PEG and lysine or histidineas antiproteases. However, upon the addition of albumin, the specificactivity decreases from 300-1200 U/mg protein to 18-24 U/mg protein.

Madaras et al. (Haemostasis 7:321-331 (1978) describe a method ofpurifying factor VIII on heparin-Sepharose and eluting with increasingNaCl concentrations. However, the factor VIII thus obtained had merelylow activity.

U.S. Pat. No. 5,252,709 describes a method of separating factor VIII,vWF, fibronectin and fibrinogen from human plasma, wherein at firstfactor VIII, vWF and fibronectin are bound to a DEAE-type ion exchangerand subsequently are eluted separately from the ion exchanger byincreasing salt concentrations.

Zimmerman et al. (U.S. Pat. No. 4,361,509) have described a method ofpurifying factor VIII, wherein factor VIII/vWF-complex is bound to amonoclonal anti-vWF antibody and factor VIII is dissociated from thecomplex by means of CaCl₂ ions. The factor VIII thus obtainedsubsequently is recovered in pure form via a further chromatographicstep, it must, however, be stabilized by the addition of human albumin.

By expressing factor VIII in recombinant cells (Wood et al. (1984),Nature 312:330-337), factor VIII could be produced by geneticengineering methods, yet only by the addition of or co-expression withvWF, a commercially usable yield of recombinant factor VIII could beobtained. To produce a pharmaceutical preparation, however, vWF isseparated from factor VIII during the purification process up to anegligible residual amount, and the purified recombinant factor VIII isstabilized with albumin (Griffith et al. (1991), Ann. Hematol.63:166-171).

For a use in the therapy of patients suffering from hemophilia A—andalso from von Willebrand syndrome, a purified factor VIII, complexedwith vWF, is desirable (Berntorp (1994), Haemostasis 24:289-297). Inparticular, it has repeatedly been emphasized that in preparationslacking vWF or having merely a low content of vWF, an extended bleedingtime and a low factor VIII:C half-life are observed in vivo.Normalisation of vWF in vivo is important for maintaining theconcentration of factor VIII in plasma both by reducing the factor VIIIelimination rate and by aiding the liberation of endogenous factor VIII(Lethagen et al. (1992), Ann. Hematol. 65:253-259).

DE 3 504 385 describes carrying out an ion exchange chromatography forpurifying factor VIII/vWF-complex, factor VIII-complex being bound viasulfate groups and eluted with citrated buffer, calcium chloride andNaCl gradient. In this instance, the factor VIII-complex is eluted fromthe carrier with a concentration of 0.5 M NaCl.

EP 0 416 983 describes the recovery of factor VIII/vWF-complex fromhuman plasma by precipitation with a combination of barium chloride andaluminum hydroxide and subsequent anion exchange chromatography onDEAE-Fractogel.

In EP 0 411 810, purification of factor VIII/vWF-complex fromcryoprecipitate is effected by means of heparin affinity chromatographyand subsequent elution of the complex with calcium chloride. A furtherdevelopment of this method is described in WO 93/22337. To removecontaminating proteins, such as fibrinogen and fibronectin, aglycine/NaCl precipitation is carried out after the elution with CaCl₂.

To purify factor VIII/vWF-complex it has also been suggested toprecipitate contaminating proteins, such as fibrinogen, with highconcentrations of amino acids, in particular glycine, to dissociatefactor VIII/vWF-complex which remains in solution by the addition of acalcium and amino acid-containing buffer, and subsequently to recoverfactor VIII and vWF separately from each other by anion exchangechromatography (WO 82/04395).

U.S. Pat. No. 5,356,878 describes the preparation of factorVIII-complex, in which contaminating proteins (fibrinogen, vitaminK-dependent factors or fibronectin) are separated by precipitation withAl(OH)₃ and PEG, factor VIII-complex is chemically virus-inactivated inthe presence of glycine and NaCl, and subsequently the non-factorVIII-complex-specific proteins are removed by gel filtration.

Hornsey et al. (Thromb. Haemost. 57:102-105 (1987)) have purified factorVIII/vWF by means of immune affinity chromatography and attained aspecific activity of 45 U of factor VIII/mg protein and 60 U ofristocetin activity/mg protein. However, the final product iscontaminated with 4% of fibrinogen and with 2% of fibronectin and withmurine antibodies detached from the carrier.

Mejan et al. (Thromb. Haemost. 59:364-371 (1988)) suggested to purifyfactor VIII/vWF-complex directly from plasma by immune affinitychromatography. The purified complex was stabilized with human serumalbumin and subsequently lyophilized. With the elution conditionsdescribed, however, a partial liberation of the antibodies from thecolumn was observed, which led to a contamination of the eluate withmonoclonal antibodies and required a second purification step forremoval of the antibodies. With their method, Mejan et al. attained anapproximately 1400-fold enrichment of the factor VIII/vWF-complex with aspecific factor VIII:C-activity and ristocetin activity of 20 U/mgprotein each, the product containing all the vWF multimers. Afterstabilization of the complex with 10 mg/ml albumin, a stability of 3-4months at −20° C. was observed. However, it has been repeatedlyemphasized that a particular difficulty in the purification of thecomplex consists in maintaining the association of the proteins, becauseboth components in the complex are unstable.

Harrison et al. (Thromb. Res. 50:295-304 (1988)) describe thepurification of factor VIII/vWF-complex by means of chromatography ondextran sulfate-agarose.

EP 0 600 480 describes the purification of factor VIII/vWF-complex bymeans of anion exchange chromatography, wherein the factorVIII/vWF-complex-containing eluate is stabilized with heparin andalbumin and optionally lysine and histidine are added as antiproteases.

Commercially available factor VIII/vWF-preparations partially have no oronly a small portion of high-molecular vWF multimers (vWF/HMW), andexhibit, particularly in dependence on the infusion time, in vivo areduction of the high-molecular vWF multimers (Lethagen et al. (1992),Ann. Hematol. 65:253-259).

The factor VIII preparations described in the prior art do mostlycontain the entire vWF multimer pattern, yet their portions of HMW-vWFand LMW-VWF vary and they exhibit so-called triplet structures,indicating a proteolytic degradation of vWF multimers, in particular ofvWF/HMW (Scott et al. (1993), Sem. Thromb. Hemost. 19:37-47, Baillod etal. (1992), Thromb. Res. 66:745-755, Mannucci et al. (1992), Blood79:3130-3137). The stability of these preparations is limited thereby.

To stabilize the preparations, either before virus inactivation or so asto obtain a storage-stable preparation, it has repeatedly beenemphasized that the addition of a stabilizer, such as albumin, isrequired.

All the factor VIII concentrates that have been obtained by purificationof the protein from human plasma or which have been in contact withbiological material from mammals furthermore bear the potential risk ofcontaining microbiological or molecular pathogens, such as, e.g.,viruses. To produce a safe preparation therefore an inactivation ofpathogenic organisms is also always necessary. Effective inactivationmethods may easily also lead to a loss of the biologic activity of thefactor VIII complex. Thus, Palmer et al. found (Thromb. Haemost.63:392-402 (1990)) that in case of heat treatment for an effective virusinactivation, an activity loss of between 17% and 30% must be reckonedwith also in the presence of a stabilizer.

It has repeatedly been emphasized that factor VIII/vWF-concentratesexhibiting an intact multimer structure possibly have a favorableinfluence on the hemorrhaging time, because they carry out the primaryfunction of vWF, i.e. platelet agglutination, and have a higher affinityto the platelet receptors glycoprotein Ib and IIb/IIIa thanlow-molecular vWF multimers (LMW-vWF) (Mannucci et al. (1987), Americ.J. Hematology 25:55-65). However, there exists the problem that thereoccurs a degradation particularly of the HMW-vWF molecules during theprocess of preparing factor VIII concentrates.

Thus, there is a need for a factor VIII-complex having a sufficientspecific activity of factor VIII:C and vWF-activity, which has animproved stability and which remains stable over an extended period oftime also without the addition of the non-factorVIII/vWF-complex-specific stabilizer.

It is thus the object of the present invention to provide a factorVIII/vWF-complex having an improved stability.

According to the invention, this object is achieved by providing afactor VIII/vWF-complex which particularly contains high-molecular vWFmultimers and which is free from low-molecular vWF molecules and fromproteolytic vWF degradation products.

The specific platelet agglutination reflects the ratio of ristocetincofactor activity and vWF antigen content. A high specific plateletagglutination activity thus indicates the specific activity of themultimers. Within the scope of the present invention it could be shownboth for plasmatic vWF (p-vWF) and for recombinant vWF (r-vWF) or factorVIII/vWF-complex, respectively that in case of a high multimerisationdegree of vWF, the specific platelet agglutination (RistoCoF/vWF:ag) issubstantially increased in comparison to low-molecular multimers.

Low-molecular p-vWF (p-vWF/LMW) and low-molecular r-vWF (r-vWF/LMW)exhibit only very low platelet agglutination.

This situation can be explained more clearly by the fact that on accountof the short vWF chain, no stable connection between several plateletswill occur. On the contrary, high-molecular (long) vWF multimers areable to connect several platelets in a stable manner.

It has been shown that both, high-molecular p-vWF (p-vWF/HMW) andhigh-molecular r-vWF (r-vWF/HMW) bind to platelets in aconcentration-dependent manner and exhibit a higher specific plateletagglutination activity than low-molecular vWF multimers.

The stable factor VIII/vWF-complex according to the invention thereforehas a specific vWF platelet agglutinating activity of at least 50 U/mgvWF:Ag.

In plasma, factor VIII/vWF in complex occurs at a molar ratio ofapproximately 1:50. It has been found that this ratio is necessary inplasma to offer a good protection against proteolytic degradation, inparticular by protein C (Vlot et al. (1995), Blood 85:3150-3157).

According to a further aspect of the present invention, the factorVIII/vWF-complex of the invention has a molar ratio of factor VIII tovWF of between 0.01 and 100. This means that the complex exhibits aratio of factor VIII molecules to vWF molecules of between 1:100 and100:1, respectively. Preferably, the molar ratio of factor VIII to vWFranges between 1:30 and 1:70, particularly preferably it is 1:50, anddue to the high portion of vWF/HMW in the complex, an optimum ratio isobtained for a protection against proteolytic degradation.

According to the invention, furthermore a stable factor VIII/vWF-complexis provided which contains high-molecular plasmatic vWF multimers withdoublet structure.

Within the scope of the present invention it has been found that fromplasma or cryoprecipitate a chromatographically purified factorVIII-complex is obtained which is comprised of vWF multimer moleculesexhibiting doublet structure. This has been surprising because vWF orfactor VIII/vWF-complex purified from plasma or cryoprecipitate had onlybeen known with a triplet structure of the vWF multimers. These tripletstructures are created by proteolytic degradation of vWF multimers andindicate an instability of the vWF multimers. Palmer et al. (Thromb.Haemost. 63:392-402 (1990)) have described that in the preparation offactor VIII concentrate from heparinized plasma and subsequent virusinactivation, the normal triplet pattern changes, and the intensity ofthe triplet band having the lowest molecular weight increases greatly,indicating an increased proteolytic degradation of the vWF multimers. Incontrast thereto, in the present invention a factor VIII/vWF is providedwhich completely lacks this vWF degradation product and whichsubstantially contains the 2 bands of the original triplet having thehigher molecular weight.

According to a further embodiment of the invention, a stable factorVIII/vWF-complex is provided which contains high-molecular recombinantvWF multimers exhibiting singlet structure. It has been found bymultimer analysis that the vWF multimers of recombinant vWF merely havesinglet structure, have a high structural integrity and are free fromany proteolytic vWF degradation products. The factor VIII-complexaccording to the invention, containing high-molecular recombinant vWFmolecules of high structural integrity therefore is very stable and isfree from low-molecular vWF multimers and vWF degradation products.

The stable factor VIII/vWF-complex according to the invention ispreferably free from plasma proteins, in particular from plasmaproteases, and it is free from fibrinogen and fibronectin. Since plasmaproteases and plasma proteins, in particular activated plasma proteins,such as protein C, factor IIa or factor IXa, vWF or factor VIII degradeproteolytically and the inventive complex is free from plasma proteins,it has an increased stability and integrity of the proteins in complex.

The complex according to the invention has an increased resistance toproteolytic degradation, and thus it is stable at room temperature,e.g., for at least 48 hours, preferably for at least 6 days, and inlyophilized form at 4° C. or room temperature for more than 2 years.

The factor VIII/vWF-complex according to the invention is so stable thatit can be provided as a virus-safe complex. Virus safety is ensured bymethod steps for treating the complex for inactivation of viruses or fordepletion of viruses, respectively.

For inactivating viruses, a heat treatment in solution or in the solid,preferably lyophilized, state is particularly suitable, and this heattreatment can reliably inactivate both lipid-enveloped andnon-lipid-enveloped viruses. The complex according to the invention is,e.g., heat treated in the solid, wet state according to EP 0 159 311.Other methods for virus inactivation comprise also the treatment withdetergents or with chaotropic substances, e.g. according to EP 0 519901, WO 94/13329, DE 44 34 538 and EP 0 131 740.

According to a further aspect of the invention, a stable, virus-safefactor VIII-complex concentrate comprising in particular high-molecularvWF multimers of high structural integrity is provided. Thehigh-molecular vWF multimers preferably have singlet or doubletstructure and are free from low-molecular vWF multimers and fromproteolytic degradation products of vWF. Surprisingly it has been shownthat the factor VIII-complex concentrate according to the invention isso stable that a treatment for virus inactivation, e.g. as describedabove, negatively affects the stability of the proteins, in particularof the high-molecular vWF multimers in the complex only marginally andthat thus the specific activity of factor VIII:C and vWF-ristocetinactivity in the factor VIII-complex or factor VIII-complex concentrateis lowered only by 10% at the most in a virus inactivation step. In thehitherto known factor VIII-complex concentrates, a loss of activity ofbetween 20 and 30% had to be reckoned with during the virusinactivation. In the neoantigen test, the factor VIII/vWF-complex of theinvention did not exhibit any changes of the antigen structure after thevirus inactivation step, which proves the stability of the proteins inthe complex. On account of the high stability of the high-molecular vWFmultimers, also a high specific platelet agglutination activity of atleast 50 U/mg vWF:Ag is ensured in the factor VIII-complex concentrateaccording to the invention.

According to a special embodiment, the stable, virus-safe factorVIII-complex-concentrate of the invention contains factor VIII and vWFat a molar ratio of factor VIII to vWF of between 0.01 and 100,preferably between 0.05 and 1. In particular, the factorVIII-complex-concentrate is free from plasma proteins, in particularfrom plasma proteases, and free from microbiological and molecularbiological pathogens.

To improve the stability of purified proteins, usually stabilizers, suchas albumin, are added. When doing so, however, the specific activity ofthe purified protein is lowered by the addition of the foreign protein.vWF is a natural component of the factor VIII-complex. It has been foundthat by the addition of high-molecular vWF multimers (vWF/HMW) to apurified factor VIII fraction from plasma or from recombinant cellcultures, the stability of factor VIII is increased. Likewise it hasbeen found that by the addition of vWF/HMW to a purified factorVIII-complex, the stability of the complex can be improved. Thus, onecan do without the addition of commonly used stabilizers. In exceptions,optionally a protease inhibitor may be added also in the course of therecovery so as to maintain the intact structure, in particular of thevWF/HMW.

According to the invention, furthermore a pharmaceutical compositioncomprising a stable factor VIII/vWF-complex according to the inventionor a virus-safe, stable factor VIII-complex-concentrate according to theinvention can be provided.

In a special embodiment, the pharmaceutical composition contains aphysiologically acceptable carrier or buffer. The formulation of thepharmaceutical preparation according to the invention may be effected ina common manner and as is known per se, e.g. by aid of salts andoptionally amino acids, but also in the presence of tensides. On thebasis of the above-described high stability of the complex, thestabilizers or protease-inhibitors commonly used may optionally also bedone without in the pharmaceutical composition.

The stable factor VIII/vWF-complex according to the invention preferablyis obtained as a highly purified product, which is obtained bychromatographic purification methods. In particular, chromatographicpurification is effected by ion exchange chromatography and/or affinitychromatography. For this, i.a. materials for anion exchange, such assynthetic carrier materials or carbohydrate-based carriers, withligands, such as DEAE, TMAE, QAE, Q or amino alkyl groups can be used,or carriers with immobilized substances which have a specific affinityto vWF can be used for affinity chromatography, respectively. Suitableaffinity materials contain heparin, e.g.

According to a further aspect of the present invention, thus a method ofrecovering stable factor VIII/vWF-complex is provided. Therein, factorVIII/vWF-complex from a protein solution is bound at a low saltconcentration to an affinity carrier, preferably a heparin affinitycarrier, and stable factor VIII/vWF-complex is recovered at a high saltconcentration. The complex preferably is bound to immobilized heparin ata salt concentration of ≦150 mM, and is eluted at a salt concentrationof a ≧200 mM and ≦300 mM. In a particularly preferred embodiment of thepresent invention, recovering of factor VIII/vWF-complex is carried outin a buffer system free from CaCl₂. In this manner factorVIII/vWF-complexes comprising low-molecular vWF molecules andhigh-molecular vWF molecules, respectively, can be selectively separatedfrom each other, and factor VIII/vWF-complex comprising in particularhigh-molecular vWF molecules can be recovered at a higher saltconcentration. Soluble mono-and divalent salts are usable for elution.Preferably, NaCl is used. Calcium salts are not suitable for elution.

Preferably, the method according to the invention is carried out on aheparin affinity chromatography column. Any carrier to which heparin canbe bound may be used for the affinity chromatography. AF-HeparinToyopearl® (a synthetic, hydrophilic polymer of large pore size based onmethacrylate) (Tosohaas), Heparin EMD Fraktogel® (a synthetic,hydrophilic polymer based on ethylene glycol, methacrylate and dimethylacrylate) (Merck) or Heparin Sepharose Fast Flows® (containing naturaldextran and agarose derivatives, respectively) (Pharmacia) have, e.g.,proved useful.

In the method according to the invention, as buffer system, a buffersolution consisting of buffer substances, in particular Tris/HCl,phosphate buffer or citrated buffer, and optionally salt, is used, whichis free from stabilizers, amino acids or other additives.

Affinity chromatography is preferably effected at a pH ranging from 6.0to 8.5, preferably at pH 7.4.

In the method according to the invention, a protein solution comprisingfactor VIII/vWF-complex, such as, e.g., a plasma fraction, acryoprecipitate or a cell-free culture supernatant derived fromtransformed cells is used. The solution may also be an enriched proteinfraction of a chromatographic method.

According to the method of the invention for recovering factorVIII/vWF-complex, a factor VIII/vWF-complex may be obtained in anefficient and simple manner, which substantially compriseshigh-molecular vWF multimers. According to this method, thus aphysiologically particularly active factor VIII/vWF-complex can beprepared in good yields and in high purity. The factor VIII/vWF-complexthus obtained is particularly characterized by a specific activity offactor VIII:C of at least 50 U/mg factor VIII:Ag and a specific vWFplatelet agglutination activity of at least 50 U/mg vWF and isparticularly free from low-molecular vWF multimers and from vWFdegradation products.

According to a further aspect of the present invention, a method ofrecovering stable factor VIII/vWF-complex is provided, in which factorVIII/vWF-complex from an impure protein solution is bound to an anionexchanger and contaminating plasma proteins at a salt concentration of≦200 mM are selectively eluted in the presence of calcium salt.Subsequently, factor VIII/vWF-complex is obtained from the anionexchanger at a salt concentration of between a ≧200 and ≦400 mM. Afactor VIII/vWF-complex substantially comprising high-molecular vWFmultimers is recovered.

To carry out the method according to the invention, e.g. a plasmafraction, a cryoprecipitate or a culture supernatant from transformedcells which is free from cells can be used as the impure proteinsolution containing factor VIII/vWF-complex.

The contaminating proteins removed by the calcium salts are, inparticular, plasma proteins, among them vitamin K-dependent factors,such as, e.g., factor II, factor IX, protein C, protein S, plasmaproteases, such as plasminogen, fibronectin or fibrinogen. Removal ofthe unspecific proteins is particularly effected with CaCl₂ as calciumsalt in the eluting agent at a concentration of between 1 mM and 15 mM,preferably of 10 mM.

It has been found within the scope of the present invention that thehitherto used method of aluminum hydroxide treatment for the separationof vitamin K-dependent proteins, fibrinogen or fibronectin is notsufficient to completely remove these proteins. By elution in thepresence of calcium chloride, however, it has been ensured that theseplasma proteins are substantially eliminated and a factorVIII/vWF-complex free from plasma proteins is recovered.

The anion exchange chromatography is preferably carried out at a pHrange of from 6.0 to 8.5, preferably at a pH of 7.4.

Elution of the factor VIII/vWF-complex bound to the anion exchangerduring the anion exchange chromatography preferably is carried out byincreasing the salt concentration.

As the anion exchanger, preferably an anion exchanger of the quaternaryamino type, in particular a Fractogel having tentacle structure, and inparticular EMD-TMAE Fractogel, is used.

Preferably, factor VIII/vWF-complex is bound to the anion exchanger at asalt concentration of ≦200 mM, and factor VIII/vWF-complex substantiallycomprising vWF/HMW is eluted at a salt concentration of ≧270 mM,preferably at a ≧350 mM. As the salts, soluble mono- and divalent saltsare usable, NaCl being preferred.

Preferably, the factor VIII complex purified by anion exchangechromatography is further chromatographically purified by affinitychromatography, preferably on immobilized heparin, in a buffer solutioncomprised of buffer substances and, optionally, salt.

In a particular embodiment of the method according to the invention, atfirst a factor VIII/vWF-complex-containing fraction recovered fromcryoprecipitate is bound to an anion exchanger, and factorVIII/vWF-complex substantially containing high-molecular vWF-multimersis eluted in enriched form after separation of the accompanyingproteins, in particular of the plasma proteins. In a furtherpurification step, the factor VIII/vWF-complex-containing eluate iscontacted with an affinity carrier comprising covalently bound heparin,the complex binding to the carrier. After removal of foreign substancesand foreign proteins by means of a suitable eluting agent, factor VIIIcomplex is eluted from the affinity carrier by means of a mono- ordivalent salt, preferably NaCl, in a buffer system.

In a further particular embodiment, the method is carried out with afactor VIII/vWF-complex obtained from recombinant cells. For this, acell-free culture supernatant from cells which co-express factor VIIIand vWF, or from co-cultured cells which have been transformed withfactor VIII on the one hand and with vWF on the other hand can be used.

With the inventive method for recovering a highly purified stable factorVIII complex, a highly purified factor VIII/vWF-complex which is freefrom antibodies, free from plasma proteins, physiologically active andfree from microbiological and molecular-biological pathogens can beobtained in a simple and efficient manner.

According to the present invention, the factor VIII/vWF-complex obtainedfrom plasma or from recombinant cells particularly containshigh-molecular vWF-multimers and is free from low-molecular vWFmolecules and vWF degradation products. If the factor VIII/vWF-complexis recovered from plasma or from cryoprecipitate, the vWF/HMW areparticularly comprised of doublet structures having high stability.Factor VIII/vWF-complex obtained from recombinant cells containshigh-molecular vWF molecules having a singlet structure, high stabilityand structural integrity.

Thus, according to the present invention it is possible by means ofdefined chromatography steps to obtain FVIII/vWF free from othercoagulation factors and from further plasma proteases. This has afavorable influence particularly on the purity and stability of thepreparation. The factor VIII/vWF-complex obtained according to theinvention thus is present in a particularly pure form. Preferably, thepurity of the complex is at least 90%, particularly preferred 95%.

Due to the fact that the obtained factor VIII/vWF-complex isparticularly stable on account of its high content of high-molecular vWFmultimers and the absence of low-molecular multimers, vWF degradationproducts, foreign proteins, such as, e.g., plasma proteins, it is notnecessarily required to add stabilizers to the purified product. Thus,the specific activity of the pure product is not lowered, e.g. whenformulating the pharmaceutical composition, and it is avoided thatpossibly impurities or infectious particles are introduced into theproduct by the addition of foreign proteins.

In a further aspect of the invention, a method of preparing a stablefactor VIII/vWF-complex is provided. In doing so, a purifiedhigh-molecular fraction of vWF molecules is added to a factor VIII orfactor VIII complex purified via a chromatographic method, whereby afactor VIII/vWF-complex is obtained having a molar ratio of factor VIIIto vWF/HMW of between 0.01 (corresponds to 1 factor VIII:100 vWF) and100 (corresponds to 100 factor VIII:1 vWF), preferably of between 0.03(1:30) and 0.07 (1:70), particularly preferred of 0.05 (1:50).

The factor VIII or factor VIII-complex purified via a chromatographicmethod may be derived from a plasma fraction, a cryoprecipitate or acell-free cell culture supernatant from transformed cells.

The high-molecular fraction of vWF molecules used for the method ofpreparing the stable complex may be derived from plasma, a plasmafraction, a cryoprecipitate or a cell-free culture supernatant fromtransformed cells. The purified high-molecular fraction of vWF moleculescontains preferably a specific platelet agglutination activity of atleast 50 U/mg vWF:Ag, particularly preferred of at least 80 U/mg vWF:Ag.

To prepare the stable factor VIII complex, a purified fractioncontaining factor VIII or factor VIII/vWF-complex is mixed with apurified high-molecular fraction of vWF molecules at a desired molarratio. For this, the content of vWF, factor VIII, the factor VIIIactivity and specific vWF activity are determined in the respectivefractions, and the desired mixing ratio is adjusted by adding therespective amount of vWF/HMW. Preferably, mixing is effected such that afactor VIII/vWF-complex forms which has a molar ratio of factor VIII tovWF of between 1:100 and 100:1, preferably of 1:50.

According to the present invention, however, also a factorVIII/vWF-complex may be prepared which has a certain ratio of specificfactor VIII:C activity to specific vWF-platelet agglutination activity.Particularly preferred is a complex having a ratio of specificacitivities of 1:1. Providing the desired mixing ratio is within thegeneral knowledge of a skilled artisan.

The stable, virus-safe factor VIII-complex as well as the factorVIII-complex concentrate provided according to the present invention maybe used both for the treatment of hemophilia A and for the treatment ofvon Willebrand syndrome. Since the factor VIII-complex-preparationaccording to the invention substantially contains high-molecular vWFmolecules, it is particularly suitable for the treatment of vWD type II.

Due to the high portion of high-molecular vWF multimers, the complex ofthe invention also has very good pharmokinetics, since the vWF/HWM havea higher specific platelet agglutination and stability in vitro and inviva and stabilize factor VIII both in vitro and in vivo. The stabilityand structural integrity of the vWF multimers in the complex yields animproved half-life of the vWF and, in particular, also of factor VIII,whereby optionally the intervals of administering the pharmaceuticalpreparation of the invention can be reduced. Thus, particularly theoccurrence of inhibitory antibodies to factor VIII in hemophilia Apatients, e.g. on account of frequent administration of factor VIIIconcentrates, is prevented.

The invention will now be explained in more detail by way of thefollowing examples as well as the drawing figures, while, however, notbeing restricted to the same.

FIG. 1 shows an SDS-PAGE analysis of the vWF multimer pattern of theindividual fractions before and after anion exchange chromatography.

FIG. 2 shows the detection of factor II in individual fractions beforeand after anion exchange chromatography and after removal of factor IIby calcium chloride elution.

FIG. 3 shows the detection of protein S in individual fractions beforeand after anion exchange chromatography and after removal of protein Sby calcium chloride elution.

FIG. 4 shows the detection of factor IX in individual fractions beforeand after anion exchange chromatography and removal of factor IX bycalcium chloride elution.

FIG. 5 shows the detection of plasminogen in individual fractions beforeand after anion exchange chromatography and removal of plasminogen byprevious lysine-Sepharose chromatography.

FIG. 6 shows SDS-PAGE analysis of the vWF multimer pattern of theindividual fractions before and after heparin affinity chromatography.

FIG. 7 shows the multimer analysis of p-vWF and r-vWF before and afterheparin affinity chromatography.

FIG. 8 shows the comparison of the binding of r-vWF/HMW and p-vWF/HMW toplatelets and the graphic representation of the added amount of vWF andplatelet-bound amount of vWF.

FIG. 9 shows the binding of p-vWF/HMW and r-VWF/HMW to platelets andmultimer analysis.

EXAMPLE 1 Purification of Factor VIII/vWF-complex by Anion ExchangeChromatography

10 g of cryoprecipitate were dissolved with 70 ml Na acetate, pH 7.0,160 mM NaCl and 50 U/ml heparin at 30° C. for 10 min and incubated forfurther 30 minutes at room temperature until its complete dissolution.The solution was cooled to 15° C. and centrifuged until undissolvedcomponents had been removed, and the supernatant was treated withaluminum hydroxide gel. The solution was bound to a Fractogel EMD TMAEanion exchanger, and the anion exchanger was washed with 180 mM NaCl and200 mM NaCl to remove foreign proteins. vWF and FVIII as a complex weresubsequently eluted by means of 400 mM NaCl. The factor VIII:C andvWF:RistoCoF activities of the starting material and of the individualfractions were determined and have been summarized in Table 1.

TABLE 1 Factor VIII:C and vWF:RistoCoF Activities of the AluminumHydroxide Supernatant and of the Fractions of the Anion ExchangeChromatography Volume vWF:RistoCoF FVIII:C Sample (ml) (mU/ml) (mU/ml)Alu supernatant 147 1060 2970 180 mM eluate 254 — — 200 mM eluate 210 —— 400 mM eluate 132 1590 2890

By means of the anion exchange chromatography, factor VIII/vwF-complexcould be obtained with an increased vWF:RistoCoF activity. To assay thevWF polymer pattern, the individual fractions of the anion exchangechromatography were analysed via SDS-PAGE (Laemmli (1970), Nature227:680-685) (FIG. 1B). The polymer pattern of the vWF in the purifiedfactor VIII/vWF-complex shows an identical band pattern and thus thesame vWF polymer composition as in the cryoprecipitate. Thus, thepurification did not lead to a proteolytic degradation of high-molecularvWF multimers in the complex.

EXAMPLE 2 Removal of Vitamin K-Dependent Proteins and Recovery of HighlyPurified Factor VIII/vWF-complex

The assays aimed at recovering a FVIII/vWF-complex free from proteasesand other coagulation factors. As described in Example 1, dissolvedcryoprecipitate was treated with aluminum hydroxide, and subsequentlypurified by means of anion exchange chromatography. To remove non-factorVIII/vWF-complex-specific proteins, 10 mM CaCl₂ were added during theelution with 180 mM NaCl. The factor VIII:C and vWF:RistoCoF activitiesof the starting material and of the individual fractions were determinedand have been summarized in Table 2.

TABLE 2 Factor VIII:C and vWF:RistoCoF Activities of the StartingMaterial and of the Individual Fractions of the Anion ExchangeChromatography Volume vWF:RistoCoF FVIII:C Sample (ml) (mU/ml) (mU/ml)Alu supernatant 146 1590 4250 180 mM eluate/ 195 — — 10 mM CaCl₂ 200 mMeluate 127 — — 400 mM eluate  83 2120 5140

The eluates were assayed for their vWF multimer pattern by means ofSDS-PAGE (FIG. 1A).

From the multimer analysis it is apparent that the 400 mM eluatecontains the high-molecular vWF multimers, and starting from thecryoprecipitate, there does not occur any loss, in particular ofhigh-molecular vWF multimers. By adding CaCl₂ ions, low-molecular vWF isseparated, and a factor VIII complex containing a higher portion ofhigh-molecular vWF molecules is obtained (FIG. 1A). Thus it isdemonstrated that by the purification method, a proteolytic degradationof the high-molecular vWF multimers is avoided and by the addition ofCaCl₂ ions it is possible to selectively remove low-molecular vWFmolecules, such as, e.g., dimers or tetramers, whereby a factor VIIIcomplex substantially comprising high-molecular vWF multimers isrecovered.

The individual purification steps containing factor VIII/vWF-complexwere analysed for the presence of vitamin K-dependent proteins beforeand during the anion exchange chromatography. To this end, individual.purification steps and the individual fractions were separated viaSDS-PAGE, the proteins were transferred onto a membrane and the vitaminK-dependent proteins were detected by means of Western blot analysis.

a. Detection of Factor II in the Individual Purification Steps

To detect factor II in the individual fractions, polyclonal serum wasused as the 1st antibody against factor II (Assera Faktor II, Stago),and detection was effected by means of alkaline phosphatase-conjugatedpolyclonal goat-anti-rabbit IgG HRP conjugate (Bio-Rad) as the 2ndantibody and subsequent chromogenic test.

From FIG. 2 it is apparent that cryoprecipitate contains factor II.Despite a preceding aluminum hydroxide precipitation, the latter iseluted from the anion exchanger (lane E) as an impurity at 400 mM NaCl(together with FVIII/vWF). It is, however, possible to selectively elutefactor II by means of 10 mM CaCl₂ (lane F), and to recover vWF/FVIII ata subsequent elution with 400 mM NaCl free from factor II (lane G).

b. Detection of Protein S in the Individual Purification Steps

To detect protein S in the purification steps, polyclonalrabbit-anti-protein S serum (Assera Protein S, Stago) was used as the1st antibody, and detection was effected with goat-anti-rabbit IgG HRPconjugate (Bio-Rad) as the 2nd antibody and subsequent chromogenic test.

FIG. 3 shows the detection of protein S in individual purification stepsbefore and after the anion exchange chromatography and after removal ofprotein S by calcium chloride elution.

From FIG. 3 it is apparent that despite a preceding aluminum hydroxideprecipitation, protein S from cryoprecipitate elutes at 400 mM NaCl(together with FVIII/vWF) from the anion exchanger (lane E) as animpurity. It is, however, possible to selectively elute protein S bymeans of 10 mM CaCl₂ (lane F), and to recover FVIII/vWF at a subsequentelution with 400 mM NaCl free from protein S (lane G).

c. Detection of Factor IX in the Individual Purification Steps

To detect factor IX in the purification steps, polyclonalrabbit-anti-factor IX serum (Assera Faktor IX, Stago) was used as the1st antibody, and detection was effected by means of goat-anti-rabbitIgG HRP conjugate (Bio-Rad) as the 2nd antibody an subsequentchromogenic test.

FIG. 4 shows the detection of factor IX in individual purification stepsbefore and after anion exchange chromatography and selective removal offactor IX by calcium chloride.

From FIG. 4 it is apparent that factor IX elutes from the anionexchanger as contamination at 400 mM NaCl (together with factorVIII-complex) despite previous aluminum hydroxide precipitation. Byadding 10 mM CaCl₂, factor IX, however, is selectively eluted (lane D),and FVIII/vWF is recovered free from factor IX (lane E) in thesubsequent elution with 400 mM NaCl.

EXAMPLE 3 Removal of Plasma Proteases and Recovery of Highly PurifiedFactor VIII/vWF-complex

The assays aimed at recovering a vWF/FVIII preparation free fromproteases and from other coagulation factors. The protease plasminogenconstitutes a substantial contamination of the cryoprecipitate. Theformer is also found in the FVIII/vWF eluate (400 mM eluate).

To remove plasminogen, cryoprecipitate, as described above, wasdissolved and treated with aluminum hydroxide gel. Subsequently, the Alusupernatant was filtered through a lysine-Sepharose gel, and from thereit was directly applied onto the anion exchanger (Fractogel EMD TMAE).FVIII/vWF was eluted from the anion exchanger with 400 mM NaCl, asdescribed before. The eluates from before and after the anion exchangechromatography were analysed for plasminogen by means of Western blot(FIG. 5). To this end, the proteins were separated by means of gelelectrophoresis using SDS-PAGE, blotted onto a membrane, and plasminogenwas detected with a polyclonal rabbit-anti-plasminogen serum (Stago) asthe 1st antibody and subsequent chromogenic test.

From the results it is apparent that by filtration on lysine-Sepharose,the protease plasminogen is selectively separated from vWF/FVIII.

EXAMPLE 4 (Presently Considered by Applicant to be the Best Mode ofCarrying out the Invention)

Heparin Affinity Chromatography of FVIII/vWF-complex

Fractogel AF EMD-heparin was used for the heparin affinitychromatography. FVIII/vWF which had been purified by anion exchangechromatography according to Example 2 (400 mM eluate) served as thestarting material. To purify FVIII/vWF via affinity chromatography, 27ml of the 400 mM NaCl Fractogel eluate were diluted 4-fold with 81 ml ofTris-HCl buffer (pH 7.4) and applied to the heparin affinity column. Thecolumn was first washed with 100 mM NaCl and subsequently, for removingunspecifically bound proteins, with 160 mM NaCl. Subsequently, thefactor VIII/vWF-complex was obtained by elution of the heparin columnwith 300 mM NaCl. The factor VIII:C and vWF:RistoCoF activities as wellas the vWF:Ag content in the starting material and in the individualfractions of the anion exchange chromatography and the heparin affinitychromatography were determined and have been summarized in Tables 4A and4B.

TABLE 4A Factor VIII:C and vWF:RistoCoF Activities of the StartingMaterial and of the Individual Fractions before and after Anion ExchangeChromatography Volume vWF:Ag vWF:RistoCoF FVIII:C Sample (ml) (μg/ml)(mU/ml) (mU/ml) Alu supernatant 75 55 1700 5260 180 mM eluate/ 64 21  43— 10 mM CaCl₂ 200 mM eluate 33  1 — — 400 mM eluate 29 137  4250 9500

TABLE 4B Factor VIII:C and vWF:RistoCoF Activities of the Startingmaterial and of the Individual Fractions before and after HeparinAffinity Chromatography Volume vWF:Ag vWF:RistoCoF FVIII:C Sample (ml)(μg/ml) (mU/ml) (mU/ml) Starting material 104 42 850 2630 160 mM eluate42 11 43 650 300 mM eluate 28 92 2550 5840

The eluates of the heparin affinity chromatography were assayed for vWFpolymer composition (FIG. 6).

From FIG. 6 it is apparent that the high-molecular portion of vWF isobtained in the 300 mM NaCl fraction. This fraction also has the highestfactor VIII:C and vWF ristocetin cofactor activities (Table 4B).

From the sum of the results it is clearly apparent that by thecombination of anion exchange chromatography and heparin affinitychromatography vWF, FVIII and also their complex can be isolated fromthe cryoprecipitate and purified. Particularly by means of heparinaffinity chromatography it is possible to separate low-molecular vWFmultimers and degradation products of vWF from cryoprecipitate.

EXAMPLE 5 Determination of the Specific Ristocetin Cofactor Activity ofPurified vWF or vWF Complex

Plasmatic vWF (p-vWF) from human cryoprecipitate and recombinant vWF(r-vWF) from the fermentation supernatant of recombinant CHO cells wereisolated by means of chromatographic methods and purified according toExample 2. By heparin affinity chromatography and elution with varioussalt concentrations, fractions of various polymerisation degrees of vWFwere isolated (according to Example 4). On the whole for p-vWF andr-vWF, fractions with low molecular weight vWF (vWF/LMW) were obtainedat 120 mM NaCl, with medium molecular weight (vWF/MMW) at 230 mM NaCland with high molecular weight (vWF/HMW) at 300 mM NaCl. These fractionswere assayed for their content of vWF:Ag by means of ELISA (AsserachromvWF®, Boehringer Mannheim), for ristocetin cofactor activity (vWFreagent, Behringwerke), their multimer structure by means of SDS-PAGEand for their platelet binding.

FIG. 7 shows the vWF polymer analysis of plasmatic vWF and ofrecombinant vWF.

It is particularly striking that before the purification of plasmaticvWF in cryoprecipitate the vWF dimers, tetramers or multimers arepresent in triplet structures. These triplet structures are degradationproducts of vWF multimers and are due to proteases present in plasma.Particularly with the vWF MMW and vWF HMW fractions, after purificationonly multimers with doublet structures are recognizable. Thus, by thechromatographic methods, vWF multimers having an altered composition andstructure as compared to vWF molecules occurring in plasma are obtained,which is due to a depletion of proteases and low-molecular vWFdegradation products.

As compared to plasmatic vWF multimers, recombinant vWF clearly showsthe presence of only one singlet band in the vWF multimers. Therecombinant vWF multimer molecules exhibit a high structural integrityand do not contain any proteolytic degradation products, as compared tothe vWF triplet structures of plasmatic vWF known from the literature.

Table 5 and Table 6 show the specific ristocetin cofactor activity(RistoCoF/vWF:Ag) for p-vWF and r-vWF.

TABLE 5 Specific Ristocetin Cofactor Activity for Various p-vWFFractions Specific RistoCoF Activity Sample (mU RistoCoF/vWF:Ag)p-vWF/LMW 3 p-vWF/MMW 10 p-vWF/HMW 56

TABLE 6 Specific Ristocetin Cofactor Activity for Various r-vWFFractions Specific RistoCoF Activity Sample (mU RistoCoF/vWF:Ag)r-vWF/LMW 1 r-vWF/MMW 6 r-vWF/HMW 41

EXAMPLE 6 Binding of p-vWF and r-vWF to Platelets

In a further test, the binding of p-vWF and r-vWF to platelets wasinvestigated. p-vWF/HMW and r-vWF/HMW, respectively, were incubated atconstant concentrations of platelets and ristocetin. Subsequently, theplatelets were separated by centrifugation (platelet sediment, boundvWF), and a supernatant (non-bound vWF) was obtained. In the startingmaterial and in the supernatant vWF:Ag and the platelet-bound amount ofvWF were determined. As a control, identical incubations were carriedout without ristocetin. These did not yield any vWF platelet bonds. Theratio of the vWF concentration in the incubation formulation and ofplatelet-bound vWF is illustrated in FIG. 8 and shows the bindingbehaviour of p-vWF/HMW and r-vWF/HMW in a direct comparison. Followingthe incubation, both the supernatants (non-bound vWF) and the vWF in theplatelet sediment (bound vWF) were assayed for their multimercomposition. The results of the multimer analysis have been assembled inFIG. 9.

EXAMPLE 7 Determining the Stability of Purified Factor VIII/vWF-complex

Fractions obtained by means of anion exchange or heparin affinitychromatography were tested for the stability of the vWF multimers aswell as for their factor VIII activity. For this purpose, the fractionsobtained in the individual purification steps were stored at −20° C., 4°C. and room temperature for a period of time of up to 60 days, and after0, 1, 3, 5, 10, 15, 20, 30 and 60 days samples were each subjected to avWF multimer analysis, a factor VIII:C and a vWF ristocetin cofactoractivity determination. There, the eluates of the anion exchangechromatography and of the heparin affinity chromatography, respectively,in which the plasma protease had been selectively removed bylysine-Sepharose or in which the vitamin K-dependent factors had beenselectively removed by calcium chloride elution showed the higheststability. Even after 30 days the vWF multimer patterns had not changedin these samples, while in the samples which had not been subjected to alysine-Sepharose chromatography or to a calcium chloride elution,respectively, the occurrence of proteolytic vWF degradation productscould be recognized in dependence on the duration of time. Formaintaining the stability of the high-molecular vWF multimers, thus inparticular the removal of plasma proteins present in the startingmaterial is necessary, since the former greatly affect or lower,respectively, the storage stability.

EXAMPLE 8 Increasing the Stability of the Purified Factor VIII Complexby the Addition of Purified High-molecular vWF Multimers

Different amounts of purified p-vWF/HMW or r-vWF/HMW were added tovarious factor VIII- or factor VIII/vWF-complex-containing fractionsobtained by chromatographic purification steps, the mixtures wereincubated at 4° C. and room temperature over a period of time of up to40 days, and the vWF multimer composition as well as the factor VIII:Cand vWF-ristocetin cofactor activities were determined after 0, 1, 5,10, 20, 25, 30, 35 and 40 days. The stability of the vWF multimers aswell as the specific ristocetin cofactor activity were the best in thoseeluates in which plasma proteins, in particular plasma proteases, hadbeen removed by a preceding chromatography. By the addition of avWF/HMW-containing fraction to the individual factor VIII or factorVIII/vWF-containing fractions or to the starting material fromcryoprecipitate, respectively, in particular in those fractions whichhad a low stability according to Example 7, an improvement of thestability could be attained. Depending on the addition of the amount ofhigh-molecular vWF multimers, the occurrence of proteolytic degradationproducts as well as a reduction of the specific activity of factor VIIIand vWF activity could be temporally retarded.

EXAMPLE 9 Virus Inactivation of Purified Factor VIII/vWF-complex and ofPurified Factor VIII/vWF-complex after the Addition of High-molecularvWF Multimers, Respectively, and Determination of Factor VIII:C andvWF-ristoCoF Activities

Individual fractions from the chromatographic purification steps as wellas fractions to which purified high-molecular vWF multimers or albumin,respectively, had been added, were subjected to a virus inactivationmethod. For this purpose, the samples were heated for 10 h at 60° C.,and subsequently again further incubated for 1 h at 80° C. Subsequently,a vWF multimer analysis and a determination of the activity of factorVIII:C and of the specific vWF platelet agglutination activity werecarried out. It has been shown that particularly those samples which hada particularly high portion of high-molecular vWF multimers and not anylow-molecular vWF degradation products after heparin affinitychromatography, and which further had a high specific ristocetincofactor activity, exhibited the least activity losses of factor VIIIand vWF. Even in samples to which a certain amount of purifiedhigh-molecular vWF multimers had additionally been added, the activityloss after the inactivation method was 10% at the most. In those samplesto which albumin had been added, the specific activity decreased uponthe addition of the stabilizer and then, once more, after theinactivation method. By this it could be demonstrated that by thepresence of vWF/HMW exclusively, or by the addition of high-molecularvWF multimers, respectively, the stability of the proteins in the factorVIII/vWF-complex can be increased substantially without substantiallyreducing the specific activity.

We claim:
 1. A method for treating hemophilia A, comprisingadministering to a patient a stable Factor VIII/vWF-complex that has aplatelet agglutination activity of at least 50 U/mg von WillebrandFactor antigen, wherein (i) the Factor VIII/vWF-complex compriseshigh-molecular vWF multimers and (ii) the composition is free fromlow-molecular vWF molecules and from proteolytic degradation products.2. The method according to claim 1, wherein the complex has a molarriatio of Factor VIII to vWF between 0.01 and
 100. 3. The methodaccording to claim 1, wherein the complex has a molar ratio of FactorVIII to vWF between 0.05 and
 1. 4. The method according to claim 1,further comprising high-molecular plasmatic vWF multimers having doubletstructure.
 5. The method according to claim 1, further comprisinghigh-molecular recombinant vWF multimers having singlet structure. 6.The method according to claim 1, wherein the high-molecular vWFmolecules have high structural integrity.
 7. The method according toclaim 1, wherein the Factor VIII/vWF-complex is free from plasmaproteins, fibrinogen and fibronectin.
 8. The method according to claim7, wherein the plasma proteins are plasma proteases.
 9. The methodaccording to claim 1, wherein the Factor VIII/vWF-complex isstorage-stable in solution.
 10. The method according to claim 1, whereinthe Factor VIII/vWF-complex has been treated to inactivate viruses. 11.The method according to claim 1, wherein the Factor VIII/vWF-complex hasbeen treated to remove viruses.
 12. A method for treating von Willebrandsyndrome, comprising administering to a patient an effective dose of astable Factor VIII/vWF-complex that has a platelet agglutinationactivity of at least 50 U/mg von Willebrand Factor antigen, wherein (i)the Factor VIII/vWF-complex comprises high-molecular vWF multimers and(ii) the composition is free from low-molecular vWF molecules and fromproteolytic degradation products.
 13. The method according to claim 12,wherein the complex has a molar ratio of Factor VIII to vWF between 0.01and
 100. 14. The method according to claim 12, wherein the complex has amolar ratio of Factor VIII to vWF between 0.05 and
 1. 15. The methodaccording to claim 12, further comprising high-molecular plasmatic vWFmultimers having doublet structure.
 16. The method according to claim12, further comprising high-molecular recombinant vWF multimers havingsinglet structure.
 17. The method according to claim 12, wherein thehigh-molecular vWF molecules have high structural integrity.
 18. Themethod according to claim 12, wherein the Factor VIII/vWF-complex isfree from plasma proteins, fibrinogen and fibronectin.
 19. The methodaccording to claim 18, wherein the plasma proteins are plasma proteases.20. The method according to claim 12, wherein the FactorVIII/vWF-complex is storage-stable in solution.
 21. The method accordingto claim 12, wherein the Factor VIII/vWF-complex has been treated toinactivate viruses.
 22. The method according to claim 12, wherein theFactor VIII/vWF-complex has been treated to remove viruses.
 23. Astable, purified Factor VIII/von Willebrand Factor (vWF)-complex thathas a platelet agglutination activity of at least 50 U/mg von WillebrandFactor antigen, wherein the complex comprises high-molecular vWFmultimers that are free from (i) low-molecular vWF molecules and (ii)proteolytic vWF degradation products.
 24. The stable FactorVIII/vWF-complex according to claim 23, wherein the complex has a molarratio of Factor VIII to vWF between 0.01 and
 100. 25. The stable FactorVIII/vWF-complex according to claim 24, wherein the molar ratio ofFactor VIII to vWF is between 0.05 and
 1. 26. The stable FactorVIII/vWF-complex according to claim 23, further comprisinghigh-molecular plasmatic vWF multimers having doublet structure.
 27. Thestable Factor VIII/vWF-complex according to claim 23, further comprisinghigh-molecular recombinant vWF multimers having singlet structure. 28.The stable Factor VIII/vWF-complex according to claim 27, wherein thehigh-molecular vWF molecules have a high structural integrity.
 29. Thestable Factor VIII/vWF-complex according to claim 23, wherein the FactorVIII/vWF-complex is free from plasma proteins, fibrinogen andfibronectin.
 30. The stable Factor VIII/vWF-complex according to claim29, wherein the plasma proteins are plasma proteases.
 31. The stableFactor VIII/vWF-complex according to claim 23, wherein the FactorVIII/vWF-complex is storage-stable in solution.
 32. The stable FactorVIII/vWF-complex according to claim 23, wherein the FactorVIII/vWF-complex has been treated to inactivate viruses.
 33. The stableFactor VIII/vWF-complex according to claim 23, wherein the FactorVIII/vWF-complex has been treated to remove viruses.
 34. A stable,virus-safe Factor VIII/vWF-complex concentrate products that has aplatelet agglutination activity of at least 50 U/mg von WillebrandFactor antigen, wherein (i) the complex comprises high-molecular vWFmultimers of high structural integrity, and (ii) the vWF multimers havesinglet or doublet structure and are free from proteolytical vWFdegradation.
 35. The stable, virus-safe Factor VIII/vWF-complexconcentrate according to claim 34, wherein the complex has a molar ratioof Factor VIII to vWF between 0.01 and
 100. 36. The stable, virus-safeFactor VIII/vWF-complex concentrate according to claim 35, wherein themolar ratio of Factor VIII to vWF is between 0.05 and
 1. 37. The stable,virus-safe Factor VIII/vWF-complex concentrate according to claim 36,wherein the aid Factor VIII/vWF-complex concentrate is free from plasmaproteins and free from microbiological and molecular-biologicalpathogens.
 38. The stable, virus-safe Factor VIII complex concentrateaccording to claim 37, wherein the plasma proteins are plasma proteases.39. A pharmaceutical composition comprising a stable FactorVIII/vWF-complex that has a platelet agglutination activity of at least50 U/mg vWG:Ag, wherein the complex comprises high-molecular vWFmultimers, and wherein the composition is free from low-molecular vWFmolecules and from proteolytic vWF degradation products.
 40. Thepharmaceutical composition according to claim 39, further comprising aphysiologically acceptable carrier.